高糖和MBL途径的补体激活对人肾小球内皮细胞表达IL-6、TNF-α的影响

Influence of High Glucose and Mannose Binding Lectin Complement Pathway Activation to IL-6 and TNF-α’s Expression by Human Renal Glomerular Endothelial Cells

目的观察高糖和甘露聚糖结合凝集素(MBL)途径补体激活对人肾小球内皮细胞(HRGEC)表达炎症因子白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)的影响,探索糖尿病肾病(DN)新的发病机制。方法体外培养正常HRGEC,随机分为正常糖组(5mmol/LD-葡萄糖)、甘露醇组(5mmol/LD-葡萄糖+25mmol/L甘露醇)和高糖组(30mmol/LD-葡萄糖),分别培养24h后,采用实时荧光定量RCR法(real-time PCR)检测各组细胞中IL-6和TNF-α mRNA的表达,采用酶联免疫吸附法(ELISA)检测细胞培养上清液中IL-6和TNF-α的蛋白浓度。另将HRGEC随机分为单纯高糖组和高糖+MBL组,两组用30mmol/LD-葡萄糖培养24h后,加入等量30%MBL缺乏的人血清,高糖+MBL组另外加入1μg/mL MBL,继续培养4h。采用流式细胞技术和免疫荧光染色分别观察细胞表面MBL、C3和膜攻击复合物(MAC)的沉积;同时检测两组IL-6、TNF-α mRNA和蛋白的表达。结果与正常糖组和甘露醇组相比,高糖组细胞IL-6、TNF-α mRNA及蛋白表达增加(P<0.05)。流式细胞技术证实,高糖+MBL组细胞表面有明显的MBL和C3的共沉积,免疫荧光染色显示细胞表面有明显MAC沉积,单纯高糖组无上述现象;与单纯高糖组相比,高糖+MBL组细胞IL-6、TNF-α mRNA以及蛋白表达增加(P<0.05)。结论高糖可以诱导HRGEC炎症因子过表达;高糖和MBL共同存在时,MBL补体途径激活,炎症因子表达明显增加,提示MBL补体途径激活作为可能的未知发病机制对DN及其炎症状态起促进作用。

Objective To investigate the effect of high glucose and mannose binding lectin（MBL） complement pathway activation’s effect on expression of Interleukin-6（IL-6） and Tumor necrosis factor-α（TNF-α） from human renal glomerular endothelial cells（HRGEC）,to explore unkown pathogenesy of diabetic nephropathy.Methods Normal HRGEC was divided randomLy into normal glucose group（5 mmol/L D-glucose）,manicol group（5 mmol/L D-glucose＋25 mmol/L manicol）and high glucose group（30 mmol/L D-glucose）.Real-time PCR was used to detect IL-6 and TNF-α mRNA expression in each group,Euzymelinked Immunosorbent Assay（ELISA） was performed to examine the protein expression of IL-6 and TNF-α in supernatant after 24 hours’ culture.HRGEC was then randomLy divided into two groups：single high glucose group and high glucose ＋ MBL group.After 24 hours’ culture with 30 mmol/L D-glucose,30% MBL deficiency human serum was added into two groups,1 μg/mL MBL was only added into high glucose ＋ MBL group,continued the culturation for another 4 hours.Flow cytometry and Immunofluorescence technique were applied to evaluate MBL,C3 and Menbrane Attacks Complex（MAC） deposition on cell surface respectively.Real-time PCR and ELISA were performed to examine mRNA and protein expression of both IL-6 and TNF-α in each group.Results Compared with normal glucose group and manicol group,the mRNA and protein expression of IL-6 and TNF-α in high glucose group were increased（P〈0.05）.Flow cytometry confirmed obvious MBL and C3 co-deposition and Immunofluorescence confirmed obvious MAC deposition on cell surface in high glucose＋MBL group.Compared with single high glucose group,the mRNA and protein expression of IL-6 and TNF-α in high glucose＋MBL group were significantly higher（P〈0.05）.Conclusion High glucose can bring inflammatory factors’ overexpression from cultured HRGEC;high glucose together with MBL can bring MBL complement pathway activation and inflammatory factors’ overexpression,this indicates that the activation of MBL complement pathway may be a potential unkown pathogenesy of diabetic nephropathy and its proinflammatory status.